Biochemical analysis of the basic helix-loop-helix transcription factor Olig2

Abstract

The basic helix-loop-helix (bHLH) transcription factors oligodendrocyte transcription factor 1 (Olig1) and Olig2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Notwithstanding these similarities, it was apparent from early on after their discovery that Olig1 and Olig2 have non-overlapping developmental functions in patterning, neuron subtype specification and the formation of oligodendrocytes. Early in development, Olig2 functions as an anti-neural bHLH transcription factor and maintains the pool of proliferating neural progenitor cells. Later on, Olig2 switches to pro-neural functions and is important for the generation of motor neurons and oligodendrocytes. Olig1 is specifically important for the maturation of oligodendrocytes. The unique biological functions of Olig1 and Olig2 reflect their distinct genetic targets, co-regulator proteins and post-translational modifications. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino terminal domain. In the phosphorylated state, Olig2 maintains anti-neural (i.e. pro-mitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma. Furthermore, the pro-mitogenic functions of phospho-Olig2 reflect, at least in part, an oppositional relationship with p53 functions. We have used genome scale ChIP-seq, RNA-seq and mass spectroscopy proteomic methods to define the molecular mechanisms whereby phosphorylation of Olig2 promotes mitosis and opposes p53-mediated responses to genotoxic damage. We show that target gene binding is not affected by Olig2 phosphorylation. However, Olig2 phosphorylation confers global gene expression changes and association with co-regulator proteins. Towards these ends, phosphorylation regulates intranuclear compartmentalization of Olig2. Finally, Olig2 meets the requirements for a suitable therapeutic target to inhibit brain tumor growth, because it (i) is essential for brain tumor growth, (ii) specific to the central nervous system and (iii) dispensable for mature brain function. Generally speaking, bHLH transcription factors require dimerization to carry out their downstream functions. Here, we describe a set of stabilized alpha-helical (SAH) mimetics that we developed to interfere with Olig2 dimerization, and thus Olig2 function. Hydrocarbon stapling successfully produced a number of alpha-helical peptides mimicking a single helix or the entire HLH domain of Olig2. However, none of the SAH OLIGopeptides specifically and potently disrupted Olig2 dimerization and subsequent DNA binding. We conclude that the HLH domain is required, but perhaps not sufficient for Olig2 dimerization. Our studies indicate that specification and differentiation of oligodendrocytes are completely independent of Olig2’s triple serine phosphorylation function in proliferation. The ability to uncouple these functions one from the other suggests an avenue to specifically target tumors within the brain while sparing normal white matter. In the fullness of time, small molecule inhibitors of Olig2 protein may thus have practical applications for glioma medicine.